Methods of producing an osteoinductive calcium phosphate material for bone grafting

ABSTRACT

The present invention relates to methods for producing biphasic calcium phosphate materials using chemical processing methods including exposure to peroxides. The resulting materials exhibit an osteoinductive needle-like surface morphology and are useful as artificial bone grafts.

FIELD OF THE INVENTION

The present disclosure generally relates to methods of producing a bonegrafting product and use of such products.

BACKGROUND

Artificial or synthetic bone can be used to repair damaged areas wherenatural regeneration may not be feasible or practical. The ability toincorporate new bone growth through osteoconductivity andosteoinductivity are important factors in artificial bone materials.Osteoconductivity is the ability to serve as a scaffold for new bonegrowth while osteoinductivity refers to the ability of graft material toinduce de novo bone growth with biomimetic substances, such as bonemorphogenetic proteins.

Recent advances in tissue engineering have produced materials such ascalcium phosphates that possess both osteoconductive and osteoinductiveproperties, thereby, providing a suitable bone grafting material.Calcium phosphates may include hydroxyapatite (HA) or beta-tricalciumphosphate (βTCP) or biphasic calcium phosphate (a combination of HA andβTCP). The osteoinductivity of calcium phosphates is a qualitativefeature and depends on various material parameters. A way to improve theosteoinductivity of calcium phosphates is through manipulation of itssurface morphology including the cultivation of HA needles or nanorodson the material surface instead of the inherent grain-like morphology ofthe post-sintering starting material.

Current techniques for inducing HA needles or nanorods and increasingosteoinductivity through surface modifications are limited in that theyrequire hydrothermal treatment at high temperatures and pressures (2-4bar and above 125° C.) and are only able to produce HA needles onβTCP/HA granules with higher concentrations of βTCP (above 80% βTCP).

SUMMARY

The present invention provides methods for preparing biphasic calciumphosphate materials with an osteoinductivity-boosting needle-like ornanorod-like surface morphology using chemical processes includingexposure to peroxides (e.g., hydrogen peroxide). Methods of theinvention are able to produce the desired needle-like or nanorod-likesurface morphology on biphasic calcium phosphate materials of any ratioincluding βTCP/HA granules with βTCP content below 80% or even 40%.Furthermore, the inclusion of chemical processing methods of theinvention avoids the need for higher heat and pressure hydrothermaltreatments while still providing the osteoinductivity-increasing surfacecharacteristics. The post-processing surface morphology of the biphasicmaterial of the invention provides increased osteoinductivity and,therefore, a superior artificial bone material suitable for a variety oforthopedic and maxillofacial treatments.

In certain embodiments, chemical processing methods include exposingbiphasic calcium phosphate materials to a peroxide (e.g., hydrogenperoxide) for a period sufficient to generate a needle-like ornanorod-like surface morphology on the material. The peroxide exposuremay take place at room temperature or temperatures higher than roomtemperature in a sealed container. This treatment can be performedeither before or after hydrothermal treatment. When applied according tothe descriptions herein, peroxide exposure can reduce the requiredtemperature, pressure, and/or time of hydrothermal treatments whiledelivering a bone graft product with high osteoinductivity. Methods ofthe invention are able to produce the desired needle-like ornanorod-like surface morphology on biphasic calcium phosphate materialsof any ratio including βTCP/HA granules with less than 80% βTCP content.

Methods of the invention are compatible with biphasic calcium phosphatestarting granules in a variety of sizes (e.g., 0.1-10 mm). Treatmentmethods may vary depending on the composition of the starting material.For example, biphasic granules consisting of 20% HA and 80% βTCP (20/80granules) can be subjected to a chemical treatment that requires soakingin hydrogen peroxide at less than 125° C. to develop the desiredneedle-like or nanorod-like surface morphology while 60/40 granules mayfirst undergo a hydrothermal treatment at temperatures above 125° C.followed by a chemical treatment such as soaking in a hydrogen peroxidesolution at temperatures less than 125° C. to replicate the desiredneedle-like or nanorod-like surface morphology.

Materials of the invention exhibit improved osteoinductivity compared topre-treatment granules and may be used to induce the formation of bonetissue in a patient alone or in combination with growth factors, cells,or other components. Biphasic calcium phosphate of the invention canhave a particle size ranging from about 0.1 mm to about 10 mm and can beused as a medical implant material or tissue scaffold. Granules of theinvention may be used in injections with or without a carrier fluid.Alternatively, the material may be formed into a composite of any sizeand shape depending on the desired application and can be sized on-siteto repair a specific bone defect.

Aspects of the invention include methods for producing a bone graftingproduct including steps of providing a granule comprising hydroxyapatite(HA) and β-tricalcium phosphate (β-TCP) and conducting a process on thegranule to produce one or more HA needle-like or nanorod-likeprotrusions from the surface of the granule, wherein the processcomprises soaking the granule in a solution comprising a peroxide. Theprocess may further include hydrothermally treating the granule prior tosoaking. The hydrothermal treatment can include autoclaving the granuleat about 140° C. The granule may be autoclaved at about 140° C. forabout 8 hours.

Steps of the method may further include drying the granule betweenautoclaving and the soaking step. Granules may comprise about 60% HA andabout 40% β-TCP. In various embodiments, the peroxide may be hydrogenperoxide (H₂O₂) and the solution may comprise about 50% H₂O₂. Thesoaking step may be performed in a sealed container for about 6 hoursaccording to certain embodiments.

In various embodiments, the hydrothermal treatment may includeautoclaving the granules prior to soaking at a temperature more than125° C. Granules may comprise about 20% HA and about 80% β-TCP. Thesolution may comprise about 30% H₂O₂. Soaking can occur in a sealedcontainer for about 4 hours.

In certain aspects, methods of the invention may include producing abone grafting product by providing a granule comprising β-tricalciumphosphate (β-TCP) and at least about 60% by weight hydroxyapatite (HA),performing a hydrothermal treatment on the granule, and soaking thegranule in a solution comprising a peroxide to thereby produce one ormore HA needle-like protrusions from the granule. The treatment mayoccur in an open container. In various embodiments, the solution maycomprise 50% hydrogen peroxide and the soaking can occur for about 6hours in a closed container.

Aspects of the invention may include a method for producing a bonegrafting product by providing a granule comprising β-tricalciumphosphate (β-TCP) and about 20% by weight hydroxyapatite (HA), andsoaking the granule in a solution comprising a peroxide to therebyproduce one or more HA needle-like protrusions from the granule. Incertain embodiments, the solution can include 30% hydrogen peroxide.Soaking can occur for about 4 hours in a closed container.

Materials of the invention may have a porosity ranging from about 50% toabout 60% with about 55%-60% consisting of micropores (less than about 3μm) and about 30%-about 35% being made up of macropores (greater thanabout 70 μm). Total pore area of treated biphasic calcium phosphate ofthe invention may be about 3 to 4 m2/g, or higher. The specific surfacearea (BET) of the materials of the invention may be more than about 2 to3 m2/g, or higher and may comprise a needle density of about 1needle/μm2 or more. Needle diameters for treated biphasic materials mayrange between about 100 and 400 nm with median diameters in a range ofabout 200 to 250 nm. As discussed in the examples below,osteoinductivity of materials of the invention was found to be increasedover that of untreated biphasic materials.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows two scanning electron micrograph (SEM) images ofpost-sintering biphasic granules consisting of 60% HA and 40% βTCP(60/40 granules).

FIG. 2 shows two SEM images of 60/40 granules after processing accordingto certain methods of the invention.

FIG. 3 shows two SEM images of post-sintering biphasic granulesconsisting of 20% HA and 80% βTCP (20/80 granules).

FIG. 4 shows two SEM images of 20/80 granules after processing accordingto certain methods of the invention.

FIG. 5 shows representative histology images for different groups of thestudy. Treatment groups had more bone formation than the control group.

DETAILED DESCRIPTION

Methods of preparing bone grafting materials consisting of biphasiccalcium phosphate are disclosed herein using chemical processing toinduce an osteoinductive needle-like surface morphology through exposureto peroxides. Specifically, the invention relates to treating biphasiccalcium phosphate granules to transform the standard post-sinteringgrain-like surface morphology into a needle-like surface morphologyshown to exhibit superior osteoinductivity. Of particular note, methodsof the invention produce the needle-like or nanorod-like surfacemorphology desired for artificial bone grafts without reliance on thehigh-temperature and pressure hydrothermal treatments of existingtechniques. Furthermore, the chemical processing methods of theinvention can generate the desired needle-like or nanorod-like surfacemorphology on granules of any ratio of calcium phosphate to apatiteincluding β-tricalcium phosphate/hydroxyapatite (βTCP/HA) granules withless than 80% or even 40% βTCP content. Previous treatment methods havebeen unable to consistently produce such a material.

Methods of the invention use chemical treatments including soaking ofbiphasic calcium phosphate granules in a peroxide solution to generatethe desired needle-like or nanorod-like surface morphology without theneed for high-temperature or pressure hydrothermal treatments. Further,such chemical processing methods have proven effective on materials witha lower proportion of calcium phosphate to apatite than is possible withcurrent techniques.

Needle-like or nanorod-like surface morphology refers to the presence ofHA needles or nanorods as shown in FIGS. 2 and 4. Grain-like surfacemorphology of the post-sintering biphasic calcium phosphate startinggranules refers to a relatively smooth surface with a substantial lackof HA needles as shown in FIGS. 1 and 3. Unless otherwise specified,percentages discussed herein with respect to HA and βTCP granulecomposition refer to percent by weight.

Methods of the invention primarily involve the chemical processing ofbiphasic calcium phosphate materials using peroxides (along with otheroptional treatments) to produce a needle-like surface morphology in thematerial. The soak time and the concentration of peroxide in thesolution can vary depending on the type of peroxide used, whether thegranules have been hydrothermally treated, and the ratio of HA to βTCPin the granules being processed. For example, in certain embodiments,hydrothermal treated granules having less than 60% βTCP content (e.g.,60/40 granules) may be soaked in a 50% hydrogen peroxide (H₂O₂) solutionfor about 6 hours while granules with higher βTCP content (e.g., 20/80granules) may be soaked in a 30% H₂O₂ solution for about 4 hours togenerate the desired needle-like surface morphology. In variousembodiments, peroxide treatment may occur in a sealed container.

Peroxides used in processing biphasic calcium phosphate materials arepreferably hydrogen peroxide but may be any compound having a peroxidegroup including peroxy acids, metal peroxides, organic peroxides, andmain group peroxides. In certain embodiments, various agents may besubstituted for the peroxide in the processing steps described above.Examples include oxidizers, NaHCO₃, Na₂HPO₄, calcium sulfate, calcite,NaCl, ammonium hydroxide, sodium hydroxide, poly(D,L-lactic-co-glycolicacid), pectin and gelatin, vesicants, cetyltrimethyl ammonium bromide,polytrimethylene carbonate, sucrose, inorganic peroxides, perchloricacid, nitric acid, perborates, periodates, peroxyacids, chlorates,chromate.

In various embodiments peroxide solutions may comprise 3%, 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% peroxide and soaking timesmay be less than about 30 minutes, less than about 60 minutes, less thanabout 100 minutes, less than about 200 minutes, less than about 300minutes, less than about 400 minutes, less than about 500 minutes, lessthan about 600 minutes, less than about 700 minutes, less than about 800minutes, less than 900 minutes or less than about 1000 minutes.

Biphasic calcium phosphate granules are used as a starting material andcan be prepared using known methods. Such granules are also commerciallyavailable in a variety of ratios including the 60/40 and 20/80 HA/βTCPcompositions primarily discussed herein.

Methods of the invention contemplate using particles of any size (e.g.,0.1 mm-10 mm) and preferably use sintered biphasic calcium phosphategranules commercially available between 0.5 mm and 2 mm in size.Particles used are preferably sintered biphasic calcium phosphategranules commercially available between 1 mm and 2 mm in size. Methodsfor preparing biphasic calcium phosphate materials through sintering andthe use of foaming and/or porogenic agents (including hydrogen peroxide)are known in the art and described, for example, in U.S. Pat. No.10,064,892 and U.S. Pat. Pub. No. 20110020419, the contents of each ofwhich are incorporated herein by reference. Additional information oncalcium phosphate granules and their use can be found in U.S. Pat. Pub.Nos. 20050260115, 20130165540, and 20090110743 as well as U.S. Pat. Nos.6,949,251, 8,460,685, and 7,942,934, the contents of each of which areincorporated herein by reference. In various embodiments, startingbiphasic calcium phosphate materials may be produced through foaming ofan aqueous slurry including a calcium phosphate powder using a foamingagent followed by drying and sintering of the resulting foamed slurry.Particle size of the starting material may be altered by milling of thesintered material to achieve the desired size range.

The ratio of calcium phosphate to hydroxyapatite in the biphasicparticles is not a limiting aspect of the invention, and the methods ofthe invention may be carried out using granules having all differentratios of calcium phosphate to apatite. In certain embodiments, 60/40HA/βTCP granules may be used as a starting material. Afterpre-processing according to the invention (or provision of commerciallyavailable material), granules have a grain-like morphology withmultidirectional interconnected porosity structure, that is about 20-30%microporous (e.g., having a pore size<10 about μm) and 50-55%macroporous. An exemplary scanning electron micrograph (SEM) for suchgranules is shown in FIG. 1. The grain-like surface morphology alongwith the microporosity of the material can be seen in the figure. Inother embodiments, granules having higher βTCP content (e.g., 20/80HA/βTCP granules) may be processed using methods of the invention togenerate the osteoinductive needle-like or nanorod-like surfacemorphology. A pre-processing image of exemplary 20/80 granules withmostly grain-like morphologies and some needles is shown in FIG. 3.

While βTCP and HA are discussed herein for purposes of illustrating themethods of the invention, application to other calcium phosphateparticles and additional apatite minerals is also contemplated. Otherapatite minerals include any calcium phosphate minerals with therepeating stoichiometric chemical formula Ca₅(PO₄)₃(OH) such ashydroxyapatite, fluoro-apatite, chloro-apatite, carbonated apatite or acalcium deficient apatite among others. Elliott, J. C., 1994. Structureand Chemistry of the Apatites and other Calcium Orthophosphates,Amsterdam: Elsevier, incorporated herein by reference.

Processing of bi-phasic calcium phosphate granules may include ahydrothermal treatment the details of which may depend on the ratio ofHA to βTCP. Hydrothermal treatment involves exposing the granules to acombination of heat, pressure, and water such as in an autoclave.Hydrothermal treatments may be performed before, after, and/or duringchemical processing with peroxide as described above. As noted above,temperature ranges for hydrothermal treatment may depend on thecomposition of the starting material and can be less than about 125° C.for granules with 60% or more βTCP content and preferably less thanabout 90° C. Pressure ranges for hydrothermal treatment are preferablybetween about 2 and 4 bar. For example, in granules having less than 60%βTCP content (e.g., 60/40 granules), hydrothermal treatment may beperformed at about 140° C. Treatment may occur, for example, by placingdry granules in an open bottle and then placing in an autoclave.Hydrothermal treatment may be performed at about 140° C. for about 600minutes in preferred embodiments but longer and shorter treatment timesare possible as well and can produce similar results. In variousembodiments, temperatures may be less than 125° C., less than 100° C.,less than 90° C., less than 75° C., or less than 50° C. Hydrothermaltreatment times may be less than about 30 minutes, less than about 60minutes, less than about 100 minutes, less than about 200 minutes, lessthan about 300 minutes, less than about 400 minutes, less than about 500minutes, less than about 600 minutes, less than about 700 minutes, lessthan about 800 minutes, less than 900 minutes or less than about 1000minutes. In certain embodiments, a thermal treatment may be used in lieuor in addition to a hydrothermal treatment. For example, dry granulesmay be treated in an autoclave without any liquid or may simply beheated at atmospheric pressure, in an oven for example.

Hydrothermal treatment can be performed using granules in any liquid.For example, granules may be submerged in an aqueous or non-aqueoussolution. Aqueous solutions can include water, hydrogen peroxide, acids,bases, etc. Non-aqueous solutions may include alcohols, etc.

Hydrothermal treatment of bi-phasic calcium with greater βTCP content(e.g., 20/80 granules), can occur at temperatures lower than 140° C.,lower than 125° C., and preferably around 90° C. or lower. Suchhydrothermal treatments can optionally be performed in an autoclave asdescribed above. Furthermore, hydrothermal treatment may be performedfor shorter time periods (e.g., about 4 hours) than for granules withlower βTCP content.

In certain embodiments, hydrothermal treatment is not required andbiphasic calcium phosphate granules may begin processing with soaking ina peroxide solution. If a hydrothermal treatment is performed, granulesmay be recovered and dried before soaking.

After chemical treatment, granules may be rinsed (e.g., with deionizedwater) and dried before final packaging or use in artificial bonegrafts. FIGS. 2 and 4 show SEM images of post-processing 60/40 and 20/80granules respectively. Notably, granules in both images exhibit a cleardevelopment of needle-like or nanorod-like surface morphology subsequentto processing methods of the invention.

While described above primarily with respect to granules, methods of theinvention may be applied to surface coatings of biphasic calciumphosphate to similarly generate the desired osteoinductive needle-likeor nanorod-like morphology for various implants or other devices.Materials of the invention may have a porosity ranging from about 50% toabout 60% with about 55%-60% consisting of micropores (less than about 3μm) and about 30%-about 35% being made up of macropores (greater thanabout 70 μm). Total pore area of treated biphasic calcium phosphate ofthe invention may be about 3 to 4 m2/g, or higher. The specific surfacearea (BET) of the materials of the invention may be more than about 2 to3 m2/g, or higher and may comprise a needle density of about 1needle/μm2 or more. Needle diameters for treated biphasic materials mayrange between about 100 and 400 nm with median diameters in a range ofabout 200 to 250 nm. Osteoinductivity of materials of the invention isincreased over that of untreated biphasic materials.

Materials prepared according to the methods described herein are usefulfor inducing bone tissue formation in patients including mammals andother organisms. Treated biphasic calcium phosphate can be used as animplant material for medical procedures such as orthopedic surgery andmaxillofacial procedures. Bone graft materials of the invention may beused as fillers or scaffolds to facilitate bone formation and promotewound healing and can be used in solid material (block) forms trimmed tofit a certain defect or may be used in a putty or paste (particulated)format. Applications of the materials prepared according to methods ofthe invention include dental implants (e.g., to restore edentulous areaof a missing tooth). In various embodiments, materials of the inventionmay be used to form large bone sections to restore skeletal integrity tolong bones of limbs in which congenital bone defects exist or to replacesegments of bone after trauma or malignant tumor invasion. Graftmaterial may also be used to fuse joints to prevent movement, repairbroken bones that have bone loss, and repair broken bone that has notyet healed.

EXAMPLES Example 1: Preparation of Osteoinductive 60/40 HA/βTCP Materialwith Needle-Like Surface Morphology

60/40 HA/βTCP granules were obtained from Biomatlante. The granules havea grain-like morphology with multidirectional interconnected porositystructure, that is 20-30% microporous (pore size<10 μm) and 50-55%macroporous. The scanning electron micrograph (SEM) for this granule isshown in FIG. 1. Microporosity is clearly visible in the granule alongwith grain-like surface structure.

The granule was further processed using the following techniques and wassubsequently imaged to see the difference in the microstructure:

First, the granules underwent a hydrothermal treatment. The treatmentinvolved placing granules (dry) contained in an open bottle and thenplacing in an autoclave. The autoclave treatment was performed at 140°C. for 600 minutes. The granules were recovered and dried at 90° C.prior to the next step.

The hydrothermally treated granules were soaked in a 50% hydrogenperoxide (H₂O₂) solution in a closed bottle for 6 hours. The granuleswere subsequently washed with deionized water and dried at 90° C. priorto imaging.

The SEM image shown in FIG. 2 demonstrates the change in microstructurefrom the pristine granules. The image demonstrates needle-likemorphology for the treated 60/40 HA/βTCP granules.

Example 2: Preparation of Osteoinductive 20/80 HA/βTCP Material withNeedle-Like Surface Morphology

The 20/80 HA/βTCP granules are also obtained from Biomatlante. Thesurface topography of the pristine granules show mostly grains with someneedles being present. The SEM image of the pristine granule is shown inFIG. 3.

The granule was further processed using the following technique and wassubsequently imaged to see the difference in the microstructure:

First, the granules were soaked in a 30% hydrogen peroxide (H₂O₂)solution in a closed bottle for 4 hours. The granules were subsequentlywashed with deionized water and then dried at 90° C. prior to imaging.

The SEM image shown in FIG. 4 shows the change in microstructure fromthe pristine granules. The image demonstrates needle-like morphology forthe treated 20/80 HA/βTCP granules.

Example 3: Osteoinductive (OI) Potential Testing

Procedure

Experimental Setup

One cc of granules of following groups were implanted in the paraspinalmuscles of skeletally mature female sheep:

1. 60/40 HA/βTCP granules 1-2 mm (control)

2. Treated 60/40 HA/βTCP granules 1-2 mm (treatment)

3. Treated 20/80 HA/βTCP granules 1-2 mm (treatment)

The sheep used in the study were greater than 2 years of age and thegranules were implanted for a period of 12 weeks in the sheep toevaluate the tissue reaction and osteoinductive property of the treatedgroups.

Surgical Procedure

Following premedication, an intravenous catheter was placed in acephalic, jugular, or lateral saphenous vein, and following anestheticinduction, the sheep was endotracheally intubated. IV fluids (LactatedRingers Solution, or equivalent balanced electrolyte solution at a rateof 2.5-10 mL/kg/hr) were given throughout the procedure. The wool overthe back was clipped, and the area was scrubbed with alternatingchlorhexidine and isopropyl alcohol for at least three cycles or untilthe sheep was clean. Once the sheep is positioned prone on the operatingtable a sterile surgical scrub was performed using chlorhexidine.Prophylactic antibiotics were administered perioperatively. Exposedareas outside of the surgical field were covered as much as possible.

A skin incision, starting at approximately L1 and continuingapproximately 10 inches caudally, was made approximately 2 inches offmidline on one side of the lumbar spine. The paraspinous muscles wasexposed and 6 intra-muscular incisions, approximately 1.5 cm in lengthand 1 inch apart, were made through the fascia and the underlying musclefibers were separated to create a pocket. One of the 3 graft materials(volume=1.0 ml) were then inserted into the muscle pockets. Thisprocedure was then repeated on the contralateral side of the spine. Onceall graft materials were implanted, soft tissues and skin were closed inlayers using absorbable suture material.

Following the surgical procedure, anesthesia was terminated and theanimal was given appropriate post-operative care prior to transferringto housing pen. The animals will be allowed free movement.

Harvest and Histological Processing

Twelve weeks after implantation, the animals were sacrificed, and thesamples were harvested with surrounding tissue. The implant sites weredissected free and removed in toto. The soft tissues were removed, andspecimens were placed in 10% neutral buffered formalin (NBF). Afteradequate fixation time in the 10% NBF, each specimen was decalcifiedwith Cal-Ex, processed in ascending ethanol solutions (70%, 80%, 90%,95% and 100%×2) and embedded in paraffin for sectioning. Sectionedspecimens were stained with H&E and each section was observed under alight microscope (Leitz Photomicroscope with an AO two-headedmicroscope) to analyze the tissue reaction and bone formation in detail.The sections were graded according to the Edwards scale

TABLE 1 Edwards scale for histological grading Grade Estimated percentof cross-sectional area under review 0 No evidence of new bone formation1 Greater than 0% up to 25% of field shows evidence of new boneformation 2 26-50% of field shows evidence of new bone formation 351-75% of field shows evidence of new bone formation 4 76-100% of fieldshows evidence of new bone formation Note: Final determination ofosteoinductive potential as either positive or negative was based solelyon the histopathology analysis of the implant sites. This determinationincluded microscopic observations of the criteria listed below (notexhaustive nor exclusive): Presence of chondroblasts/chondrocytesPresence of osteoblasts/osteocytes Presence of cartilage/osteoidPresence of new bone Presence of bone marrowResults

A total of twelve (12) samples were implanted intra-muscularly into theparaspinal muscles of skeletally mature female sheep. There were threegroups evaluated in this study with four (4) samples per each group.After 12 weeks, samples were retrieved with their surrounding tissue. Noinflammation was seen in any of the explants. Bone formation wasobserved in varying quantities in all the groups of the study. As perEdwards scale, the osteoinductive potential of the groups were in thefollowing order: Treated 20/80 HA/βTCP>Treated 60/40 HA/βTCP>60/40HA/βTCP. Average osteoinductivity scores across four implants for eachgroup are shown in Table 2.

TABLE 2 Osteo Inductivity Results Group Osteoinductivity Score 60/40HA/β-TCP (control) 1.3 Treated 60/40 HA/β-TCP (Treatment) 1.7 Treated20/80 HA/β-TCP (Treatment) 1.8

The representative histological images for the control and two treatmentgroups are presented in FIG. 5. As shown in Table 2 and FIG. 5,significantly more bone formation was observed in the treatment groupsthan the control group. The new bone is represented by the darkeststaining toward the center of the two treated groups and nearly absentfrom the control group.

Example 4: Scanning Electron Microscopy

Procedure

Scanning electron microscopy in the secondary electron mode (SEM; JEOL)was used to evaluate the surface topography of the starting granules andthe treated granules. After hydrothermal treatment, the diameter of 100formed needles was measured, and median values were calculated. Allmeasurements were performed with the tool ‘length measurement’ in ImageJ(v1.43u, NIH, USA) using the SEM scale bar as reference.

Results

The SEM images demonstrated that the grain-like surface as seen onpretreated materials was successfully transformed to needle-like ornanorods-like surface after the peroxide treatment. The diameter datafor Treated 60/40 HA/βTCP and Treated 20/80 HA/βTCP is presented inTable 3.

TABLE 3 SEM surface characterization of treated groups Sample NeedleDiameter (nm) Median Diameter (nm) Treated 60/40 HA/βTCP 260 ± 100 258Treated 20/80 HA/βTCP 235 ± 128 196

Example 5: BET Surface Area

Procedure

For BET surface area by gas physisorption, the analysis was conductedusing the Micromeritics TriStar II instrument. Briefly, a representativealiquot of sample (approximately 2 g) was added to a sample cell with0.5″ neck. To remove moisture from the sample surfaces and pores, thesample was degassed under vacuum at 40° C. for 16 hours prior toanalysis. Analysis was conducted at 77.35K using nitrogen gas as theadsorbate. Saturation pressure of nitrogen was measured by theinstrument throughout the experiment. Adsorption and desorption processwas allowed to equilibrate at each relative pressure (P/PO) for 20seconds. The surface area was calculated from 5 adsorption points in theP/PO range of 0.05-0.20 using the BET method.

Results

BET surface area are mentioned in Table 4. The data suggests that theneedle-like or nanorod-like formations on the granule surface lead toincrease in their specific surface area.

TABLE 4 BET Specific Surface Area measurements before and aftertreatment for 60/40 HA/βTCP and 20/80 HA/βTCP BET Specific SampleTreatment Surface Area (m²/g) 60/40 HA/βTCP Pristine 2.06 Treated 3.1820/80 HA/βTCP Pristine 2.20 Treated 2.32

Example 6: Mercury Intrusion Porosimetry

Procedure

For pore size distribution and porosity by mercury intrusionporosimetry, the analysis was conducted using the Micromeritics AutoPoreV instrument. Briefly, a representative aliquot of sample (approximately0.7 g) was added to a calibrated 5 cc powder penetrometer with a stemvolume of 1.131 cc. To remove moisture from the sample surfaces andpores, the sample was evacuated on the instrument at room temperature toa target pressure of 30 μm Hg. After further applying vacuum for 5minutes, the penetrometer bulb was filled with mercury at about 0.5psia. Pressures of up to around 50,000 psia were applied to forceintrusion of mercury into the void space in the sample. Equilibration ateach pressure step was monitored by rate of intrusion (0.050 μL/g/sec).Baseline errors are compensated by a blank analysis using the samepenetrometer under similar analysis conditions. The pore sizedistribution results in the range of interest (0.02 to 450 μm) werecalculated using the Washburn equation, assuming cylindrical porestructure, 140° for the mercury contact angle, and the appropriatemercury density based on the temperature at the time of analysis.

Results

The mercury intrusion porosimetry results are presented in Table 5. Thedata suggests that there is slight increase in microporosity and thetotal pore area from the untreated to the treated groups of the samecomposition.

TABLE 5 BET Specific Surface Area measurements before and aftertreatment for 60/40 HA/βTCP and 20/80 HA/βTCP Macro- Micro- Total TotalPore pores >70 pores <3 Porosity Area Sample Treatment μm (%) μm (%) (%)(m2/g) 60/40 HA/ Pristine 41.04 52.03 55.96 2.649 βTCP Treated 35.1256.95 52.42 4.181 20/80 HA/ Pristine 37.51 51.05 58.15 2.81 βTCP Treated33.15 54.47 58.51 2.934Needle DensityProcedure

Needle density on the granule was determined by counting the number ofneedles visible on a 10000×SEM image (window size 1132.81 um²). Thedensity was calculated per um².

Results

The needle density on treated 60/40 HA/βTCP and treated 20/80 HA/βTCPgranules were greater than 1 needle/um².

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

What is claimed is:
 1. A method for producing a bone grafting product,the method comprising: providing a granule comprising hydroxyapatite(HA) and β-tricalcium phosphate (β-TCP); conducting a process on thegranule to produce one or more HA needle or nanorod protrusions from thesurface of the granule, wherein the process comprises soaking thegranule in a solution comprising an oxidizing agent.
 2. The method ofclaim 1, wherein the process further comprising prior to the soakingstep, hydrothermally treating the granule.
 3. The method of claim 2wherein the hydrothermal treatment comprises autoclaving the granule atabout 140° C.
 4. The method of claim 3 wherein the granule is autoclavedat about 140° C. for about 8 hours.
 5. The method of claim 4 furthercomprising drying the granule between autoclaving and the soaking step.6. The method of claim 2 wherein the granule comprises about 60% HA andabout 40% β-TCP.
 7. The method of claim 6 wherein the oxidizing agent ishydrogen peroxide (H₂O₂).
 8. The method of claim 7 wherein the solutioncomprises about 50% H₂O₂.
 9. The method of claim 8 wherein the soakingstep occurs in a sealed container for about 6 hours.
 10. The method ofclaim 2 wherein the hydrothermal treatment comprises autoclaving thegranule at a temperature less than 125° C.
 11. The method of claim 10wherein the granule comprises about 20% HA and about 80% β-TCP.
 12. Themethod of claim 11 wherein the peroxide is hydrogen peroxide (H₂O₂). 13.The method of claim 12 wherein the solution comprises about 30% H₂O₂.14. The method of claim 13 wherein the soaking step occurs in a sealedcontainer for about 4 hours.
 15. A method for producing a bone graftingproduct, the method comprising: providing a granule comprisingβ-tricalcium phosphate (β-TCP) and at least about 60% by weighthydroxyapatite (HA); performing a hydrothermal treatment on the granule;and soaking the granule in a solution comprising a peroxide to therebyproduce one or more HA needle or nanorod protrusions from the granule.16. The method of claim 15 wherein the hydrothermal treatment occurs inan open container.
 17. The method of claim 15 wherein the solutioncomprises 50% hydrogen peroxide and the soaking occurs for about 6 hoursin a closed container.
 18. A method for producing a bone graftingproduct, the method comprising: providing a granule comprisingβ-tricalcium phosphate (β-TCP) and about 20% by weight hydroxyapatite(HA); and soaking the granule in a solution comprising a peroxide tothereby produce one or more HA needle or nanorod protrusions from thegranule.
 19. The method of claim 18 wherein the solution comprises 30%hydrogen peroxide.
 20. The method of claim 19 wherein soaking occurs forabout 4 hours in a closed container.